ABSTRACT
With improvements in biophysical approaches, there is growing interest in characterizing large, flexible multi-protein complexes. The use of recombinant baculoviruses to express heterologous genes in cultured insect cells has advantages for the expression of human protein complexes because of the ease of co-expressing multiple proteins in insect cells and the presence of a conserved post-translational machinery that introduces many of the same modifications found in human cells. Here we describe the preparation of recombinant baculoviruses expressing DNA ligase IIIα, XRCC1, and TDP1, their subsequent co-expression in cultured insect cells, the purification of complexes containing DNA ligase IIIα from insect cell lysates, and their characterization by multi-angle light scattering linked to size exclusion chromatography and negative stain electron microscopy.
Subject(s)
DNA Ligases , DNA-Binding Proteins , Animals , DNA Ligase ATP/genetics , DNA Ligase ATP/metabolism , DNA Ligases/chemistry , DNA-Binding Proteins/metabolism , Humans , Insecta/metabolism , Poly-ADP-Ribose Binding Proteins , X-ray Repair Cross Complementing Protein 1 , Xenopus Proteins/metabolismABSTRACT
R-loops are three-stranded nucleic acid structures with both physiological and pathological roles in cells. R-loop imaging generally relies on detection of the RNA-DNA hybrid component of these structures using the S9.6 antibody. We show that the use of this antibody for imaging can be problematic because it readily binds to double-stranded RNA (dsRNA) in vitro and in vivo, giving rise to nonspecific signal. In contrast, purified, catalytically inactive human RNase H1 tagged with GFP (GFP-dRNH1) is a more specific reagent for imaging RNA-DNA hybrids. GFP-dRNH1 binds strongly to RNA-DNA hybrids but not to dsRNA oligonucleotides in fixed human cells and is not susceptible to binding endogenous RNA. Furthermore, we demonstrate that purified GFP-dRNH1 can be applied to fixed cells to detect hybrids after their induction, thereby bypassing the need for cell line engineering. GFP-dRNH1 therefore promises to be a versatile tool for imaging and quantifying RNA-DNA hybrids under a wide range of conditions.
Subject(s)
DNA/metabolism , Inverted Repeat Sequences , RNA, Double-Stranded/metabolism , Recombinant Fusion Proteins/metabolism , Ribonuclease H/metabolism , Staining and Labeling/methods , Antibodies/chemistry , Antibodies/metabolism , BRCA1 Protein/antagonists & inhibitors , BRCA1 Protein/genetics , BRCA1 Protein/metabolism , Cloning, Molecular , DNA/chemistry , DNA/ultrastructure , DNA Helicases/antagonists & inhibitors , DNA Helicases/genetics , DNA Helicases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Gene Expression , Genes, Reporter , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HeLa Cells , Heterocyclic Compounds, 4 or More Rings/chemistry , Heterocyclic Compounds, 4 or More Rings/metabolism , Humans , Multifunctional Enzymes/antagonists & inhibitors , Multifunctional Enzymes/genetics , Multifunctional Enzymes/metabolism , Nucleic Acid Hybridization , Optical Imaging/methods , Protein Binding , RNA Helicases/antagonists & inhibitors , RNA Helicases/genetics , RNA Helicases/metabolism , RNA, Double-Stranded/chemistry , RNA, Double-Stranded/ultrastructure , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Recombinant Fusion Proteins/genetics , Ribonuclease H/geneticsABSTRACT
Mechanistic studies in DNA repair have focused on roles of multi-protein DNA complexes, so how long non-coding RNAs (lncRNAs) regulate DNA repair is less well understood. Yet, lncRNA LINP1 is over-expressed in multiple cancers and confers resistance to ionizing radiation and chemotherapeutic drugs. Here, we unveil structural and mechanistic insights into LINP1's ability to facilitate non-homologous end joining (NHEJ). We characterized LINP1 structure and flexibility and analyzed interactions with the NHEJ factor Ku70/Ku80 (Ku) and Ku complexes that direct NHEJ. LINP1 self-assembles into phase-separated condensates via RNA-RNA interactions that reorganize to form filamentous Ku-containing aggregates. Structured motifs in LINP1 bind Ku, promoting Ku multimerization and stabilization of the initial synaptic event for NHEJ. Significantly, LINP1 acts as an effective proxy for PAXX. Collective results reveal how lncRNA effectively replaces a DNA repair protein for efficient NHEJ with implications for development of resistance to cancer therapy.
